猪精液常温保存效果观察

为研究不同保存条件对猪精液常温保存效果的影响,观察精子的活力和有效保存时间之间的关系,采用常温下对精液添加抗生素、不同精液稀释比例、光照、振荡、混入尿液等,每隔12 h 显微镜检测一次精子活力的方法,以筛选常温下猪精液保存方式。结果显示,在精液保存过程中加入抗生素时,精液有效保存时间和精子活力明显高于未加抗菌药物的稀释液,其中加入庆大霉素的保存效果最好;精液稀释比例为1∶1～1∶2时保存效果最好;光照及剧烈振荡条件下,精液活力下降很快;混入尿液很容易使精子死亡。上述研究结果表明,常温下猪精液的理想保存方式是,在保证精子密度的前提下,精液与稀释液比例为1∶1～1∶2;同时在精液稀释液中添加广谱抗生素;保存过程中应避免光照、振荡、尿液污染等的影响。     

猪精液稀释剂中抗微生物药物的选择

精子在采集和保存过程中,不可避免地会受到一些污染物的影响，临床上常用青霉素、链霉素或者庆大霉素添加于精液稀释剂中．在精液的稀释保存过程中可以提高对有害细菌的杀灭能力。从而提高精子的保存活性。目前随着抗生素在临床的广泛应用。细菌耐药性问题越来越普遍，过去常用的抗生素效果变得不明显。     

稀释倍数对湖羊精液低温保存效果的影响

为探究稀释倍数对湖羊精液4℃保存的影响，本实验使用3只1～2岁体况良好的公羊，将采集的精液稀释2、5、10、15、20、25倍后，检测4℃保存过程中精子活率、活力、运动速率以及质膜完整性和顶体完整性。结果表明：保存3～5 d,10倍稀释保存的精子活率和活力显著高于其他各组；保存1～4 d,25倍高倍稀释的精子活力下降最快，且显著低于其他组；保存1～3 d,10倍稀释保存的精子曲线速率（VCL）和路径速率（VAP）显著高于20和25倍稀释组；保存期间，10倍稀释保存的精子质膜完整性最高，且在第4天显著高于其他组；保存3～5 d,5和10倍稀释保存的精子顶体完整性显著高于15～25倍稀释组。因此，在本实验条件下，5～10倍为湖羊精液4℃保存的最适宜稀释倍数，过低或者过高的稀释倍数都不利于湖羊精液4℃保存。     

不同处理对猪精子活率、顶体完整率、DNA含量及精清酶活性的影响

对猪全精进行稀释、保存、冷冻、冷休克处理,测定相关指标.结果表明,精液1:1稀释后,在室温(25℃)保存过程中,精子活率、顶体完整率逐渐下降,精清中GOT活性持续升高,ALP活性变化不明显,LDH活性首先于保存的第24h升高,以后下降.精液1:10稀释后,精子活率急剧降低,顶体完整率没有显著性变化;随室温保存时间的延长,两者呈下降趋势,到室温保存的第24h,除GOT活性升高外,其它两种酶活性变化不明显.精液经冷冻解冻、冷休克处理后,精子活率、顶体完整率大幅度下降,精清中GOT、LDII、ALP活性升高,并随冷休克处理时间的延长而加剧;稀释后再进行冷休克处理,各指标变化幅度减小.本试验结果还表明,猪精液DNA含量为3.14mg/10~9精子,其与精子密度呈正相关(r=0.893),试验中各种处理均未引起精子DNA含量明显变化.     

死精子对猪精液17℃液态保存的影响

为揭示17℃液态保存过程中猪精子质量和功能持续下降的机制，本试验采集9头在役的不同品种公猪（长白、大白和杜洛克）的精液48份，使用BTS溶液等体积稀释后分为3份，其中的两份分别使用反复冻融（方法 A）和低渗处理（方法 B）方法杀死精子并离心获得稀释液A和B，将另一份精液样品离心分离精浆（Seminal Plasma,SP）和精子。分别使用不同体积比例稀释液A:精子（1:1、4:1和19:1），稀释液B:精子（1:1、4:1和19:1）,SP:精子（1:1和4:1）和BTS:精子（4:1）重悬离心后的猪精子，以SP和BTS重悬的精液及原精液样品作为对照，所有精液样品于17℃下保存3 d。结果表明：随着保存时间的延长，所有稀释液和稀释倍数处理均损伤了精子活率和活力（P<0.01）及质膜完整性（P<0.05）。稀释液A比B引起的精子脂质过氧化水平更低（P<0.05）。5倍稀释精子，稀释液B的精子活力较BTS组降低（P<0.05），稀释液B引起的精子细胞内活性氧族（ROS）水平比稀释液A更高（P<0.05），而精液总抗氧化物（TAC）水平更低（P<0.05）...     

家禽精液稀释液的发展

家禽精液是一种高度浓缩精子液,其容量小,为了增加精液的体积,提高单位体积中授精母禽数,常用稀释液对精液进行稀释。稀释后的精液,精子分布均匀,运输方便。在保存期间,稀释后的精液能维持和保护精子,最大     

牛冷冻精液的保存及使用

精液冷冻保存是将精液进行特殊处理，保存在超低温条件下以达到长期保存的目的。通常采用液氮（－１９６℃）和干冰（－７９℃）保存。其最大优点是可长期保存，使用不受时间、地域以及提高种用公牛的利用率。１稀释液配方１２％的蔗糖液７５mL、甘油５mL、卵黄液２０mL。２稀释方法将经过检查合格的精液（活力０．６以上、精子密度中等）按１∶２～５倍稀释液进行稀释，稀释后应保证每个颗粒精液中所含精子数不少于３０００～４０００万个，解冻后呈直线前进的精子数３冷冻在装有液氮的容器上置一铜纱网，距液氮面２cm左右，温度维持在－…     

第四讲猪人工授精

第八节精液的稀释一、精液稀释的目的猪的精液如果不经过稀释,在体外最多保存半小时,活力很快下降,而且很快失去受精能力。这是因为精清促进精子运动,缩短精子体外保存时间(见图1)。精液的稀释就是用稀释液降低精子的密度,为精子提供营养、缓冲物质,并维持适当的渗透压和pH,以利于精液的保存。因此要求原精液应尽快稀释。     

波尔山羊精液保存影响因素的探讨

为了推行波尔山羊的统一供精,延长波尔山羊人工授精过程中精液的保存时间,我们在波尔波尔山羊改良过程中,在丰县梁寨镇合作种羊场进行了波尔山羊精液的高倍稀释、常温保存、冷冻技术及授精效果的系列试验,对波尔山羊精液保存若干影响因素进行了探讨,现将结果报告如下。1精液的处理波尔山羊精液采集后,往往由于处理不当而缩短精子的保存时间。为此,我们选用5只公波尔山羊,采取每周采精5次,连续采精8周,采用4种不同处理方法,以寻求延长精子保存时间的有效途径。1.1不同稀释倍数对精子存活的影响取活力0.7～0.8,密度约20亿/mL,PH值6.4～6.6的…     

会泽常用稀释粉对种猪精液保存效果的影响

为了筛选出效果较好的精液稀释粉,试验选择会泽县常用的三种稀释粉对大约克公猪精液按一定比例进行稀释,采用MS-FX-101A型种猪精液质量检测系统检测稀释精液的精子活力、密度,姬姆萨染色后镜检精子畸形率。结果表明:三种稀释粉对常温下种猪稀释精液的精子活力影响较大,对畸形率的影响较小;保存5 d后,Ⅰ号(日本产稀释粉)稀释精液的精子活力为(58.89±2.27)%,Ⅱ号(比利时产稀释粉)稀释精液的精子活力为(57.78±0.38)%,Ⅲ号(韩国产稀释粉)稀释精液的精子活力为(38.78±6.83)%;Ⅰ号与Ⅱ号之间差异不显著(P0.05),Ⅰ号、Ⅱ号与Ⅲ号之间差异极显著(P0.01);Ⅲ号稀释精液的精子畸形率略低,但三种稀释精液精子的畸形率差异不显著(P0.05)。说明Ⅰ、Ⅱ号稀释粉的精液保存效果较好,较适合猪人工授精站推广使用。     

Effect of heterologous seminal plasma and semen extenders on motility of frozen-thawed ram spermatozoa

Ram seminal plasma increases the fertility of frozen-thawed ram spermatozoa deposited into the cervix. The aim of the current study was to compare the effect of ram seminal plasma to that of bull seminal plasma, dog prostatic fluid, protein-free TALP TrilEq (Triladyl with 0.5 mt of Equex STM paste added to each 100 mt) and heat-treated skim milk on longevity and percentages of progressively motile and aberrantly motile frozen-thawed ram spermatozoa. Three ejaculates from each of 6 rams were extended in TrilEq, pooled and frozen in straws as a single batch per ram. One hundred and eight straws (3 straws from each ram for each fluid) were thawed in random order. Once thawed, a straw was emptied into a tube with 0.85 ml of the appropriate fluid at 37 degrees C and kept at that temperature for 6 h. Motility was assessed at x200 magnification immediately (time zero) and 2, 4 and 6 h after thawing. Progressive motility decreased from each time to the next (P < 0.05) and was 39.0 % (0 h), 26.0 % (2 h), 19.6 % (4 h) and 12.6 % (6 h); SEM 1.24, n = 108 for each group. Ram seminal plasma resulted in higher progressive motility than bull seminal plasma, lower than milk, and similar to the other fluids. Ram seminal plasma resulted in lower aberrant motility than protein-free TALP and similar aberrant motility to other fluids. The effect of ram seminal plasma and dog prostatic fluid was very similar. The effect of ram seminal plasma on the fertility of frozen-thawed ram spermatozoa deposited into the cervix is not due an exceptionally beneficial effect on the motility of spermatozoa.     

Effect of staining and freezing media on sortability of stallion spermatozoa and their post-thaw viability after sex-sorting and cryopreservation

Sex‐sorted, frozen–thawed stallion spermatozoa remain out of reach of commercial horse breeders because of the low efficiency of the sex‐sorting process and unacceptable fertility rates after insemination. Two experiments were designed to test the effects of alternative staining and freezing media to improve the viability of sex‐sorted frozen–thawed stallion spermatozoa. Experiment 1 compared two freezing media, INRA 82^® and a modified lactose‐ethylenediaminetetraacetic acid (EDTA), for the cryopreservation of sex‐sorted stallion spermatozoa. No significant differences between the two freezing media could be identified, suggesting that both cryodiluents would be suitable for incorporation into a sex‐preselection protocol for stallion spermatozoa. Experiment 2 compared Kenney’s modified Tyrode’s (KMT) and Sperm TALP (Sp‐TALP) as the staining and incubation medium for stallion spermatozoa prior to sex‐sorting. A significant increase in the percentage of acrosome‐reacted spermatozoa occurred after staining and incubation in the clarified Sp‐TALP compared with KMT. As no improvements in sorting rates were achieved using Sp‐TALP, it was concluded that stallion sorting protocols could include KMT as the staining and incubation medium while either INRA 82^® or lactose‐EDTA could be employed as a cryodiluents.     

Effect of relaxin on the motility, acrosome reaction and utilization of glucose of fresh and frozen-thawed bovine spermatozoa

The study was conducted to investigate the effect of relaxin on motility, acrosome reaction (AR), viability and utilization of glucose in fresh and frozen‐thawed bovine spermatozoa. Both semen samples were washed twice through centrifugation (5 min at 600 g), and preincubated for 1 h at 39°C for swim up. The swim‐up separated spermatozoa were resuspended in a sperm Tyrode's albumin lactate pyruvate (Sp‐TALP) medium containing 0 (control) and 40 ng/mL porcine relaxin and incubated for 0–6 h. Sperm motility was determined on the basis of movement quality examined by a phase contrast microscope. Sperm viability and AR were evaluated by using the triple staining technique. The incorporation and oxidation of ¹⁴C‐glucose was assessed by a liquid scintillation counter. Motility was improved (P < 0.05) in both fresh and frozen‐thawed spermatozoa by the addition of relaxin to the Sp‐TALP medium, whereas relaxin showed no significant effect on viability in either fresh or frozen‐thawed spermatozoa. The percentage of AR increased (P < 0.05) when fresh or frozen‐thawed spermatozoa were incubated with relaxin. In contrast, the incorporation and oxidation of ¹⁴C‐glucose increased (P < 0.05) in both kinds of spermatozoa incubated with relaxin. Thus the results demonstrated that the addition of relaxin to the Sp‐TALP medium increased the motility, AR and utilization of glucose in fresh and frozen‐thawed bovine spermatozoa.     

Effect of Albumin and Polyvinyl Alcohol on the Vitality, Motility and Acrosomal Integrity of Canine Spermatozoa Incubated in vitro

Sperm culture media used for in vitro fertilization (IVF) procedures are important factors concerning the viability, motility and acrosomal integrity of spermatozoa. The aim of this study was to investigate the effects of three different sperm diluting media, tissue culture medium (TCM‐199), sperm culture medium (Sp‐TALP) and human tubular fluid (HTF) supplemented with varying concentrations of bovine serum albumin (1, 4 and 6%) or polyvinyl alcohol (0.8%) on the acrosomal integrity, motility and viability of canine spermatozoa. Ejaculates collected from four dogs were diluted in all media and spermatozoa were separated from seminal plasma by the swim‐up technique. Sperm progressive motility was assessed using a phase contrast microscope. Viability and acrosomal integrity were evaluated using a dual stain technique (Giemsa–Trypan blue). The results demonstrated that the number of live canine spermatozoa was similar in culture media supplemented or not supplemented with macromolecules. A minimal concentration of albumin (1%) in the three media showed similar effects on vitality, motility and acrosomal integrity, as had higher concentrations (4 and 6%). The percentage of acrosome‐intact spermatozoa was markedly higher after HTF (94.1%) than after TCM‐199 (70.1%) or Sp‐TALP (71.0%) without supplementation. It is concluded that serum bovine albumin, irrespective of the concentration, preserved sperm viability and function, and HTF is the most suitable medium for preserving the acrosome in canine spermatozoa prepared for in vitro manipulation through short incubation.     

Development of Procedures for Sex-sorting Frozen–Thawed Bovine Spermatozoa

Dairy bull sperm may be sex‐sorted, frozen and used to artificially inseminate heifers with acceptable fertility if the herd is well‐managed. One drawback to the technology is that donor bulls must be located within a short distance of the sorting facility in order to collect semen, which limits the number of bulls from which sorted sperm are available. A successful method used to overcome this limitation in sheep is sex‐sorting from frozen–thawed semen and refreezing for artificial insemination. This technique is attractive to the dairy industry, and therefore a series of three experiments was designed to investigate the optimal methods to prepare, sex‐sort and re‐freeze frozen–thawed bovine sperm. Sperm were prepared for sorting by density gradient separation in either PureSperm^® or BoviPure?, followed by staining in one of three diluents (Androhep^®, Bovine Sheath Fluid + 0.3% BSA or TALP buffer). Sperm were sorted and collected into Test yolk buffer, and frozen in an extender containing 0, 0.25, 0.375 or 0.5% Equex STM Paste. Frozen–thawed sperm were better orientated (p = 0.006) and had fewer damaged membranes (8.7 ± 0.6% vs 19.5 ± 2.4%; p = 0.003) after centrifugation in PureSperm^® rather than BoviPure? gradients. Sperm orientation (p < 0.05) and motility (69.9 ± 3.0 vs 55.6 ± 4.0; p < 0.001) were highest after staining in Androhep^® rather than in TALP buffer. Sperm were more motile (58.2 ± 4.7 vs 38.7 ± 3.5; p < 0.001) and had better acrosome integrity (74.3 ± 2.9 vs 66.8 ± 2.0; p < 0.001) after freezing in an extender containing 0.375% Equex STM Paste than in extender without Equex. Hence, a protocol has been developed to allow frozen–thawed bull sperm to be sex‐sorted with high resolution between the sexes, then re‐frozen and thawed with retention of motility and acrosome integrity.     

Evaluation of Spermatological Parameters Used to Predict the Fertility of Frozen Bull Semen

Kjxstad, H., E. Ropstad and K. Andersen Berg: Evaluation of spermatological parameters used to predict the fertility of frozen bull semen. Acta vet. scand. 1993,34,299-303.– Post-thaw motility, velocity and acrosome integrity of frozen semen were determined in 18 bulls with varying fertility (average non-return rates: 71.3 (± 2.8) - range: 65.2-75.7). Five semen straws were investigated from each bull. The average values for sperm motility (percentage motile spermatozoa), sperm velocity (graded from 0-3) and acrosome integrity (proportion of spermatozoa with intact acrosome) were 67.5%, 2.5 and 79.3%, respectively. Significant correlations were found between sperm motility and velocity, but not between sperm motility and acrosome integrity. Both sperm motility and velocity were significantly related to bull fertility. It was concluded that of the post-thaw semen characteristics investigated in this study these 2 parameters provided a reliable basis for prediction of bull fertility.     

Morphological changes and sperm decapitation as a cause of fertility disorders in breeding stock

Decapitation of spermatozoa was recorded in a young boar as a consequence of epididymitis and in a young dog (mastiff) and a young bull (Brown Swiss breed) as a congenital cause of the subfertility and sterility of the affected animal. Decapitation of spermatozoa as a consequence of an inflammation led to the sterility of the breeding male and affected 52 to 68% of the spermatozoa. The motility of the spermatozoa was reduced considerably (10 to 20%) and the flagella were not observed to move without the heads. In cases of congenital decapitation of spermatozoa, sperm motility in the dog and bull was reduced, but the flagella were observed to move without the heads. Decapitation affected 15 to 42% of the spermatozoa and most frequently it was accompanied by a narrowing of head base and by the presence of a protoplasmic drop, located proximally. Further developmental changes, affecting the flagella of the sperms, were observed in the dog. The mentioned morphological changes led to infertility in the dog and to a substantial reduction of fertility in the bull. After natural mating of the bull with a superovulated cow, the ten eggs obtained included four blastocysts, two were degenerated, and four were unfertilized.     

Validation and Usefulness of the Sperm Quality Analyzer (SQA II-C) for Bull Semen Analysis

In this study, an upgrade version of the Sperm Quality Analyzer (SQA), the SQA-IIC was tested for the assessment of bull semen quality. In Expt 1, the device showed good repeatability of measurements within and between capillaries, as evidenced by the low coefficients of variation (CVs; < 13%) at concentrations between 35 and 705 x 10(6) spermatozoa/ml. In Expt 2, 10 semen concentrations (1-1000 x 10(6)/ml) were stored in HEPES TALP for 48 h at room temperature. A time-dependent decrease in sperm motility index (SMI) values was noticed. SMI values increased linearly with increasing sperm concentrations, but remained constant around 500, corresponding to a concentration of approximately 50 x 10(6)/ml. For sperm concentrations below 50 x 10(6)/ml, SMI values were highly correlated with concentration (p < 0.05) and with semen parameters, expressing the overall semen quality (p < 0.05; Expt 3). In Expt 4, a correlation of only 0.44 (p < 0.05) between SMI values of frozen-thawed semen samples of 35 bulls and the corrected 56-day non-return rate (56dNRRc) was found. Prediction of the 56dNRRc based on the SMI value of a semen sample was inaccurate. The present study indicates that the SQA-IIC is suitable for a rapid screening of bull semen diluted to a concentration of approximately 50 x 10(6)/ml. Furthermore, the device seems inappropriate for fertility prediction.     

Insemination with Sex Sorted Fresh Bovine Spermatozoa Processed in the Presence of Antioxidative Substances

Flow cytometrically sex sorted spermatozoa are reduced in their fertilizing capacity, particularly when stored either in cooling extender or after freezing in liquid nitrogen. So far, preservation methods for sorted spermatozoa have differed only marginally from procedures used for unsorted semen. In the present study, a TRIS extender was modified to balance major cell damage caused by the sorting process and by liquid storage of the sorted spermatozoa. The new extender, containing a combination of antioxidants (AO) and bovine serum albumin (BSA), significantly increased the lifespan and fertilizing capacity of sex sorted spermatozoa. No significant differences were observed between unsorted controls and sorted samples for motility and status of sperm membranes as tested by fluorescein-isothiocyanat-peanut agglutinin/propidium iodide (FITC-PNA/PI). Acrosome integrity of spermatozoa was significantly better when semen was stored at 15 degrees C for 24 and 48 h in an extender containing AO with or without BSA as compared with controls (p < 0.05). There were no significant differences, in pregnancy rates of heifers inseminated at a natural oestrus, between unsorted controls (16/24, 66.7%) and both sorted groups (AO + BSA: 18/31, 58.1% and AO-BSA: 12/22, 54.5%). Additionally, it was shown for the first time that artificial insemination (AI) with liquid sexed bull spermatozoa stored for 72 h after sorting can result in pregnancy rates similar to AI with non-sorted semen.     

Retained Functional Integrity of Bull Spermatozoa after Double Freezing and Thawing Using PureSperm® Density Gradient Centrifugation

The main aim of this study was to compare the motility and functional integrity of bull spermatozoa after single and double freezing and thawing. The viability and morphological integrity of spermatozoa selected by PureSperm density gradient centrifugation after cryopreservation of bovine semen in two commercial extenders (Experiment 1) and the function of bull spermatozoa before and after a second freezing and thawing assisted by PureSperm selection (Experiment 2) were examined. On average, 35.8 +/- 12.1% of sperm loaded onto the PureSperm density gradient were recovered after centrifugation. In Experiment 1, post-thaw motility and acrosome integrity were higher for spermatozoa frozen in Tris-egg yolk extender than in AndroMed, whether the assessments were made immediately after thawing [80.4 +/- 12.7 vs 47.6 +/- 19.0% motile and 78.8 +/- 8.3 vs 50.1 +/- 19.5% normal apical ridge (NAR), p < 0.05] or after preparation on the gradient (83.3 +/- 8.6 vs 69.4 +/- 15.9% motile and 89.5 +/- 7.2 vs 69.1 +/- 11.4% NAR, p < 0.05). For semen frozen in Tris-egg yolk extender, selection on the PureSperm gradient did not influence total motility but significantly improved the proportion of acrosome-intact spermatozoa. After the gradient, both the total motility and percentage of normal acrosomes increased for spermatozoa frozen in AndroMed (Minitüb Tiefenbach, Germany). In Experiment 2, there was no difference in sperm motility after the first and second freeze-thawing (82.9 +/- 12.7 vs 68.8 +/- 18.7%). However, the proportion of acrosome-intact spermatozoa was significantly improved by selection through the PureSperm gradient, whether measured by phase contrast microscopy (78.9 +/- 9.7 vs 90.4 +/- 4.0% NAR, p < 0.05) or flow cytometry (53.4 +/- 11.7 vs 76.3 +/- 6.0% viable acrosome-intact spermatozoa, p < 0.001). The improvement in the percentage of spermatozoa with normal acrosomes was maintained after resuspension in the cooling extender and cooling to 4 degrees C (88.2 +/- 6.2) and after re-freezing and thawing (83.6 +/- 6.56% NAR). However, flow cytometric assessment of the sperm membranes revealed a decline in the percentage of viable spermatozoa with intact membranes after the second freezing and thawing compared with after gradient centrifugation (76.3 +/- 6.0% vs 46.6 +/- 6.6%, p < 0.001) to levels equivalent to those obtained after the first round of freeze-thawing (53.4 +/- 11.7% viable acrosome-intact spermatozoa). Sperm movement characteristics assessed by computer-assisted analysis were unaffected in the population selected on the PureSperm gradients but declined after cooling of the selected and extended spermatozoa to 4 degrees C. There was no further change in these kinematic measurements after the cooled spermatozoa had undergone the second round of freeze-thawing. These results demonstrate that bull semen can be frozen and thawed, followed by a second freeze-thawing cycle of a population of spermatozoa selected by PureSperm, with retained motility and functional integrity. This points to the possibility of using double frozen spermatozoa in bovine artificial insemination programmes and to the potential benefits of PureSperm density gradient centrifugation for the application of cryopreserved bull spermatozoa to other biotechnological procedures such as flow cytometric sex sorting followed by re-freezing and thawing.